Summary

Zebrafish represents a powerful in vivo model for phenotype-based drug discovery to identify clinically relevant small molecules. By utilizing this model, we evaluated natural product derived compounds that could potentially modulate Notch signaling that is important in both zebrafish embryogenesis and pathogenic in human cancers. A total of 234 compounds were screened using zebrafish embryos and 3 were identified to be conferring phenotypic alterations similar to embryos treated with known Notch inhibitors. Subsequent secondary screens using HEK293T cells overexpressing truncated Notch1 (HEK293TΔE) identified 2 compounds, EDD3 and 3H4MB, to be potential Notch antagonists. Both compounds reduced protein expression of NOTCH1, Notch intracellular domain (NICD) and hairy and enhancer of split-1 (HES1) in HEK293TΔE and downregulated Notch target genes. Importantly, EDD3 treatment of human oral cancer cell lines demonstrated reduction of Notch target proteins and genes. EDD3 also inhibited proliferation and induced G0/G1 cell cycle arrest of ORL-150 cells through inducing p27KIP1. Our data demonstrates the utility of the zebrafish phenotypic screen and identifying EDD3 as a promising Notch antagonist for further development as a novel therapeutic agent.

Notes

Acknowledgements

We would like to thank all the donors of Cancer Research Malaysia, a non-profit research organization. We are committed to an understanding of cancer prevention, diagnosis and treatment through a fundamental research program. We are thankful to Dr Deming Chao (Universiti Putra Malaysia) for providing the pHes1(467)-luc and the pCS2 Notch1 ΔEMV-6MT construct and for suggestions on Notch signaling. We would also like to thank Tan Jun Hao and Ong Yong Yong for their assistance as well as Dr Lau Beng Fye for his guidance and suggestions in cell cycle analysis.

Compliance with ethical standard

Conflict of interest

The authors declare they have no conflict of interest.

Funding

This work was supported by Cancer Research Malaysia and funding received from Ministry of Education (MOE) of Malaysia and Universiti Putra Malaysia for financial support under the research grant no. ERGS/1/11/STG/UPM/01/24.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. Procedures for animal experimentation were approved by UKM animal ethical committee (UKMAEC) ethical procedures (CARIF/2015/KAZUHIDE/25-MAR./673-JUNE-2015-DEC-2017).